Separation and preparation of N -glycans based on ammonia-catalyzed release method

The study of carbohydrates requires large amounts of glycans. N -Glycans can be synthesized but generating large quantities of N -glycans with diverse structures remains difficult. In this study, we aimed to obtain large amounts of glycans using an optimized procedure. Two types of reductive N -glycans were released from chicken egg albumin (ovalbumin) and soy protein using an ammonia catalysis method and labeled with benzenesulfonyl hydrazide (BSH). After preliminary separation by preparative HPLC, N -glycan-BSH components were de-labeled separately and reducing N -glycans were recovered. The de-labeled reducing N -glycans were derived with different labeling reagents and further separated and purified with two/multi-dimensional HPLC for various studies. We selected the bifunctional reagent 2-amino- N -(2-aminoethyl)-benzamide (AEAB) as a labeling reagent combined with C18 column for two-dimensional HPLC separation. A total of 21 and 8 N -glycan-AEAB conjugates were obtained from ovalbumin and soy protein, respectively. A reactive primary alkylamine of N -glycan-AEAB conjugates can be effectively immobilized on microarray surfaces, allowing for subsequent functional studies of glycans.